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Experimental Neurology 162, 328–342 (2000)doi:10.1006/exnr.1999.7327, available online at http://www.idealibrary.com on

Decrease in Striatal Enkephalin mRNA in Mouse Models ofHuntington’s Disease

Liliana Menalled,* Hadi Zanjani,* Larami MacKenzie,* Ahrin Koppel,† Ellen Carpenter,† Scott Zeitlin,‡and Marie-Francoise Chesselet*

*Department of Neurology and Mental Retardation Center and †Neuropsychiatric Institute, University of California at Los Angeles Schoolof Medicine, Los Angeles, California 90095; and ‡Department of Pathology, Columbia University, New York, New York 10032

Received August 11, 1999; accepted November 17, 1999

Huntington’s disease is a devastating progressiveneurodegenerative illness characterized by massiveneuronal loss in the striatum. It is caused by the pres-ence of an expanded CAG repeat in the gene encodinghuntingtin, a protein of unknown function. We haveexamined the expression of neurotransmitters andother antigens present in striatal neurons with immu-nohistochemistry, and the level of expression ofmRNAs encoding enkephalin, substance P, and glu-tamic acid decarboxylases with quantitative in situhybridization histochemistry, in the striatum of twomouse models of Huntington’s disease: transgenic an-imals expressing exon 1 of the human huntingtin genewith 144 CAG repeats and “knock-in” mice containinga chimeric mouse/human exon 1 with 71 or 94 CAGrepeats inserted by homologous targeting. Althoughthe transgenic (but not the knock-in) mice were pre-viously shown to display prominent huntingtin- andubiquitin-containing nuclear inclusions in striatalneurons, in situ nick translation followed by emulsionautoradiography did not reveal any DNA damage instriatum or cortex in these mice. Immunolabeling forcalbindin D 28K, enkephalin, substance P, glutamicacid decarboxylases (Mr 65,000 or 67,000, GAD65 and

AD67), somatostatin, choline acetyltransferase,arvalbumin, and glial fibrillary acidic protein wereemarkably similar in transgenic, knock-in, and wild-ype mice. Both transgenic and knock-in mice, how-ver, showed a marked decrease in the level of expres-ion of enkephalin mRNA in striatal neurons withoutignificant decreases in mRNAs encoding substance P,AD65, or GAD67. The data indicate that decreasedxpression of enkephalin mRNA may be an early signf neuronal dysfunction due to the Huntington’s dis-ase mutation. © 2000 Academic Press

Key Words: striatum; Huntington’s disease; enkepha-lin; transgenic mice; neurodegenerative disease; insitu hybridization; in situ nick translation; immuno-histochemistry.

3280014-4886/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

INTRODUCTION

Huntington’s disease (HD) is an autosomal domi-nant neurodegenerative disorder characterized by mo-tor disturbance, cognitive loss, and psychiatric mani-festations. The disease typically starts in midlife andsymptoms gradually worsen over a course of 15 to 20years until death. Between 3 and 10% of HD patientsexhibit symptoms before age 20 (32). Chorea is themost common involuntary movement in adult onsetHD; however, rigidity predominates in the juvenile-onset form (62).

The histopathological hallmark of HD is a pro-nounced atrophy of the caudate nucleus and putamen,subcortical structures that are part of the basal gangliaand together form the striatum (63). Striatal neuronsare not uniformly affected in HD. It has been consis-tently shown that GABA-ergic projecting neurons de-generate, whereas interneurons containing acetylcho-line, somatostatin/neuropeptide Y/NADPH-diapho-rase, and GABA/parvalbumin are relatively spared inpatients with HD (22–24, 33).

The striatal efferent neurons that degenerate in HDcan be divided in two main classes based on theirprojections and peptide content (13). Neurons contain-ing the neuropeptide enkephalin project to the externalpallidum; neurons containing the neuropeptides sub-stance P and dynorphin project primarily to the inter-nal pallidum and the substantia nigra, although theysend axon collaterals to the external pallidum as well(41). This distinction among efferent neurons led to thenotion of two separate striatal output systems: an “in-direct pathway” by way of the external pallidum andsubthalamic nucleus and a “direct pathway” projectingdirectly to the internal pallidum and substantia nigra(3, 5)

This distinction between output pathways of the stri-atum may be particularly relevant to HD. Indeed, im-munohistochemical studies have shown that presymp-tomatic carriers, as well as patients at the early stagesof the disease, have a greater decrease in enkephalin in

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329STRIATAL ENKEPHALIN mRNA IN HUNTINGTON’S DISEASE MODELS

the external segment than in substance P in the inter-nal segment of the globus pallidus (2, 51, 56). Thissuggests that enkephalinergic neurons of the striatummay be more vulnerable to the mutation causing HDthan substance P/dynorphin-containing neurons (2, 4).It is not clear, however, whether this difference inpeptide immunoreactivity is related to a difference inpeptide synthesis, storage, or release or to the selectivedeath of enkephalinergic neurons early in the disease.

The identification of the mutation causing HD as anunstable expansion of CAG repeats within the firstexon of the gene encoding huntingtin has made possi-ble the generation of mouse models of HD (36). Theseanimal models are particularly valuable to examineearly changes in neuronal functions that may precedeovert neuronal loss. Indeed, such studies are rarelypossible in humans because most brains available atautopsy already show advanced stages of degeneration.Accordingly, little is known about the early effects ofthe mutation. These effects are likely to be criticallyimportant to explain how the mutation causes the de-layed, progressive, and selective neuronal loss charac-teristic of the disorder (63). They may also be critical tounderstanding the mechanisms of the symptoms ofHD. Indeed, data from the few brains that have beenexamined at early stages of the disease suggest thatpsychiatric disorders and even chorea may occur in theabsence of detectable neuronal loss (Vonsattel grade 0),suggesting that they are due to neuronal dysfunctionrather than neuronal loss (50).

In an effort to identify the early effects of the HDmutation on striatal neurons, we have examined thelevel of expression of mRNAs encoding enkephalin,substance P, and the GABA synthesis enzymes glu-tamic acid decarboxylases (Mr 67,000, GAD67, and Mr

65,000, GAD65) in the striatum of two mouse models ofHD. The first model is a transgenic mouse expressingexon 1 of the human HD gene with 144 CAG repeats(47). This number of CAG repeats is far greater thanthe normal range in mice (7 repeats) and is larger thanthat observed in most humans with HD (approximately36–60 for adult onset, greater than 60 for juvenileonset). These transgenic mice display progressive mo-tor dysfunction (9, 47) and widespread nuclear inclu-sions, containing the transgene and ubiquitin, in brainneurons (16). Previous studies have revealed a de-crease in the expression of dopaminergic and glutama-tergic receptors in the striatum of these mice (10).These alterations preceded neuronal loss, which wasobserved only shortly before the death of the animals(S. Davies, personal communication). In addition, anearly loss of the serotonin metabolite 5-hydroxyindoleacetic acid and a delayed loss of serotonin and dopa-mine were seen in the striatum of these mice (52).

In the second mouse model that we have examined, achimeric mouse/human exon 1 containing 71 or 94CAG repeats was inserted into the mouse huntingtin

gene by homology recombination (45, 46). These micedid not develop neuronal nuclear inclusion up to 8months of age (45, 46). In both mouse models, however,striatal and cortical neurons exhibited an increasedsensitivity to stimulation of NMDA receptors. This in-dicates that the mutation alters the cellular propertiesof neurons that are affected in HD, even in the absenceof nuclear inclusions (46).

In the present study, we further compared thesetransgenic and “knock-in” mice by examining the levelof expression of mRNAs encoding enkephalin, sub-stance P, and glutamic acid decarboxylase in the stri-atum with quantitative in situ hybridization histo-hemistry. In addition, the pattern of expression ofntigens expressed in striatal neurons was examinedualitatively with immunohistochemistry, and theresence of DNA damage was assessed in the trans-enic mice by in situ nick translation.

MATERIALS AND METHODS

Animals

All procedures were carried out in accordance withthe USPHS Guide for Care and Use of LaboratoryAnimals and were approved by the Institutional Ani-mal Care and use Committee at UCLA. Two types ofmice expressing the HD mutation and their respectivecontrols have been used in this study.

Transgenic mice. Breeding pairs from the line R6/2transgenic for exon 1 of the human HD gene carrying141–156 CAG repeat expansions (47) were obtainedfrom Jackson Laboratories (B6CBA-Tg(Hdexon1)62)nd the line established at UCLA. Breeding groupsere housed in triads with one transgenic male and

wo wild-type females. Offspring were identified usingCR protocols as previously described to identify ani-als which carried the transgene as well as their wild-

ype littermates (47). Nontransgenic littermates weresed as controls. Mice were approximately 3 months ofge at the time of the experiment. Mice from two dif-erent litters were used for immunohistochemistry and

ice from five different litters were used for the in situnick-translation and in situ hybridization experiments.

Knock-in mice. Knock-in mice were generated byone of us (Scott Zeitlin, Columbia University) as de-scribed in detail elsewhere (46). Mice carrying a tar-geted insertion of human exon 1 expanded CAG re-peats were generated by gene targeting as described inLevine et al. (46). For these experiments, gene target-ing in embryonic stem cells (Sv129) was used to ex-change the normal mouse huntingtin exon 1 with achimeric mouse/human exon 1 that contained a stretchof expanded CAG repeats and the polyproline repeatsfrom a human HD patient. Correctly targeted cellswere injected into C57BL/6J host blastocysts and usedto produce chimeric animals which were screened for

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germ-line transmission of the insertion. Animals car-rying the insertion were identified by PCR (46). Thestretches of CAG repeats are interrupted by a CGGtriplet (encoding an arginine) in position 42. Overex-pression experiments in vivo and in vitro indicate thathis mutation does not interfere with the ability of therotein to form aggregates (S. Suhr, M.-C. Senut, and. Gage, personal communication). Immunoblotting

Wu et al., in preparation) and immunohistochemistry45, 46) indicate that the levels of expression of hun-ingtin are similar in mice homozygote for the muta-ion and in wild-type mice.

Mice homozygotes for the mutated allele (either 71 or4 CAG repeats) were compared to aged-matched wild-ype siblings of the 71 CAG repeat mice. Mice used forhe in situ hybridization experiments were z3.9onths of age, and immunohistochemistry was per-

ormed in mice 3 and up to 8 months of age. In situybridization experiments were performed in micerom three to four litters for each group.

reparation of Tissue and in Situ HybridizationHistochemistry

Mice were killed by decapitation and brains wereapidly removed and frozen in isopentane in dry ice.rains were stored at 280°C until sectioning. Ten-icrometer-thick sections were cut on a Leica CM 1800

ryostat (Deerfield, IL), thaw mounted on gelatin-oated slides, and kept at 280°C until the day of thexperiment.The cDNA for preproenkephalin was generously pro-

ided by Dr. S. L. Sabol (NIMH, Bethesda, MD). A70-bp transcript from the gene encoding preproen-ephalin A was isolated from a rat striatal library (65).he cDNA for b-preprotachykinin (the precursor ofubstance P) was kindly provided by Dr. J. E. KrauseWashington University, St. Louis, MO). This cDNAonsists of a 560-bp sequence isolated from a rat li-rary (42). The cDNAs encoding GAD65 and GAD67ere generously provided by Dr. A. J. Tobin (UCLA,os Angeles). The GAD67 cDNA consists of a 2.3-kbequence isolated from a cat occipital cortex library39). The GAD65 cDNA consists of a 2.4-kb sequencesolated from a rat hippocampal library (21). The plas-

ids containing the cDNAs were linearized with theppropriate restriction endonucleases, and 35S-radiola-

beled RNA probes were transcribed following standardprotocols (12, 14). Briefly, the synthesis mixture con-sisted of 10mM UTP; 2.5mM [35S]UTP (1000 Ci/mmol;NEN/Du Pont, Boston, MA); ATP, CTP, and GTP inexcess; the appropriate RNA polymerase; dithiothrei-tol; ribonuclease inhibitor (RNAsin); and 2mM linear-ized DNA containing the insert. The GAD67 andGAD65 RNA probes were subjected to partial alkalinehydrolysis into 100- to 200-bp fragments for increasedtissue penetration (12, 14). The probes were extracted

in phenol/chloroform/isoamyl alcohol and precipitatedovernight in ethanol at 280°C and their lengths veri-

ed with denaturing formaldehyde gel electrophoresis48).

In situ hybridization histochemistry was performedccording to published protocols (12, 14). All solutionsnd buffers were prepared with distilled water treatedith 1% diethylpyrocarbonate (DEPC; Sigma) to in-ibit RNase activity and autoclaved. Experimentsere performed on coronal sections through the stria-

um (A-P: 1.10 mm from the Bregma) based on thetlas of Franklin and Paxinos (26). Sections wererought to room temperature from 280°C under atream of cool air, postfixed in 3% paraformaldehydeontaining 0.02% DEPC, acetylated, and dehydrated.ections were incubated with 3–5 ng probe (400,000pm/ng) in humid chambers at 50°C for 3.5 h. Posthy-ridization treatments included three washes in 50%ormamide in 23 SSC (0.3 M NaCl/0.03 M sodiumitrate) at 52°C and a 30 min incubation in 100mg/ml

RNase A (Sigma) at 37°C. After an overnight rinse in23 SSC containing 0.05% Triton X-100, sections weredehydrated in graded ethanols, defatted in xylene, anddesiccated. All tissue sections were processed for filmautoradiography with 3H-Hyperfilm (Amersham, Ar-lington Heights, IL). The sections were subsequentlycoated with Kodak NTB3 emulsion diluted 1:1 with 300mM ammonium acetate for microscopic analysis. Testslides and strips of film were developed at regularintervals to determine optimal exposure time (15–40days), i.e., when specific labeling was robust but notsaturating based on autoradiographic standards. Bothfilms and emulsion autoradiograms were developed inKodak D-19 developer (for the emulsion sections thedeveloper was at 14°C) and fixed in Kodak Rapid Fix.The sections were lightly counterstained with hema-toxylin and eosin and mounted with Eukitt mountingmedium (Calibrated Instruments, Hawthorne, NY).The specificity of labeling for each of the RNA probesused in this study has been characterized in previousstudies (14, 49, 55) and was confirmed in each experi-ment by examining the anatomical distribution of thelabeled cells.

Quantification of in Situ HybridizationHistochemistry Results

The relative levels of expression of the different mR-NAs studied were measured on film autoradiograms(12). Films were digitized with a Hewlett–PackardScanJet 4c, and the image was visualized and analyzedwith a Power Macintosh 9500 equipped with the publicdomain NIH Image program version 1.6. Gray valueswere converted into optical densities with the help of astandard curve generated from Kodak autoradio-graphic standards (Kodak, Rochester, NY). Opticalmeasurements were taken separately for the right and

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331STRIATAL ENKEPHALIN mRNA IN HUNTINGTON’S DISEASE MODELS

the left dorsolateral striatum. These measurementswere averaged to determine a single value per section.Values obtained in two sections were averaged for eachanimal and the resulting number was used to calculategroup means.

In Situ Nick Translation

In situ nick translation (ISNT), a histological methodor the detection of DNA strand breaks based on these of labeled nucleotides and DNA polymerase I, waserformed on tissue sections according to a modifica-ion (8) of the protocol described by Iseki and Mori (37).ections were rapidly air-dried, postfixed in 3% para-

ormaldehyde in phosphate buffered saline (PBS; pH.4) for 5 min, and washed in PBS and 23 SSC, suc-essively. Next, incubation in a 50% formamide/23SC solution was carried out at 52°C for 30 min. Tissueections were acetylated in 0.1 M triethanolamine (pH.0) at room temperature and incubated in Buffer A (50M Tris–HCl (pH 7.5), 5 mM MgCl2, 10 mM b-mer-

captoethanol) containing 2mM [35S]UTP for 1 h at 37°C.The nick-translation buffer (29ml) consisting of BufferA with nonradiolabeled and 35S-labeled dTTP (12.5mCi/ml; New England Nuclear, Boston, MA); 0.01 mM eachdGTP, dCTP, and dATP; and 100 U/ml DNA polymer-ase I (Promega, Madison, WI) was then applied tosections. The concentrations of radiolabeled and colddTTP were calculated for each experiment to achieve afinal specific activity of 0.1mCi/ml and a total final con-centration of 2mM. Slides were covered with glass cov-erslips, placed in humid chambers, and incubated for30 min at 37°C. For negative controls, sections wereincubated in the same buffer without DNA polymeraseI. The reaction was stopped by rinses in ice-cold 50 mMTris–HCl (pH 7.4) followed by dehydration and defat-ting, and sections were desiccated until processed forautoradiography. Tissue sections were exposed to 3H-Hyperfilm (Amersham) and, following development,were coated with Kodak NTB3 emulsion (VWR Scien-tific, Bridgeport, NJ) as described previously (12). Sec-tions were counterstained with hematoxylin and eosin.

Immunohistochemistry

Mice were perfused with 4% paraformaldehyde in0.1 M PBS, pH 7.2, and the brain was removed andpostfixed in the same fixative overnight at 4°C, washedwith PBS, cryoprotected in 30% sucrose, and cut into20-mm-thick sagittal sections on a cryostat. Tissue sec-tions were washed extensively in PBS and treated withH2O2 (0.6–1%) in PBS containing 10% methanol for 20min to destroy endogenous peroxidase activity. Non-specific sites were blocked for 1 h at room temperaturein PBS containing 10% normal goat serum, 2% bovineserum albumin, and 0.3% Triton (incubation buffer).Sections were then incubated overnight at 4°C in theprimary antibody diluted in the incubation buffer. The

following primary antibodies and dilutions were used:met-enkephalin, substance P, and somatostatin 28polyclonal antibodies raised in rabbit, 1:500 (INCSTARCorp., Stillwater, MN); GAD67 polyclonal antibodyraised in rabbit, 1:2000 (donated by Dr. A. Tobin,UCLA); GAD65 monoclonal antibody, 1:100 (donatedby Dr. A. Tobin, UCLA); choline acetyltransferase an-tibody raised in rabbit, 1:500 (Chemicon International,Inc., Temecula, CA); calbindin D 28K monoclonal an-tibody, 1:200 (Sigma); parvalbumin monoclonal anti-body, 1:800 (Sigma); and glial fibrillary acidic proteinpolyclonal antibody raised in rabbit, 1:50 (ZYMEDLaboratories, Inc., South San Francisco, CA).

After the incubation in the primary antibody, sec-tions were washed in PBS and incubated for 2 h atroom temperature in the same buffer containing either1:200 biotinylated goat anti-rabbit antibody or 1:200biotinylated goat anti-mouse antibody (Vector Labora-tories, Burlingame, CA). After washes in PBS, sectionswere reacted with the avidin–biotin Vectastain kit(Vector Laboratories) according to the manufacturer’sinstructions, with diaminobenzidine as chromogen inthe presence of H2O2 (DAB Peroxidase Substrate Tab-let Set; Sigma). Some sections were counterstainedwith thionin. All sections were dehydrated, defatted inxylene, and mounted with Eukitt (Calibrated Instru-ments, Hawthorne, NY).

Statistical Analysis and Data Presentation

Only sections processed concurrently for in situ hy-ridization histochemistry were compared quantita-ively. All statistical analyses were done on absolutealues with the STATVIEW 5121 interactive statistics

and graphics package (version 1.0, Abacus Concepts,Calabasas, CA). Comparisons between the transgenicmice and their nontransgenic littermates were madewith an unpaired, two-tailed Student t test. Compari-sons between the knock-in mice (94 CAG repeats and71 CAG repeats) and the corresponding control micewere made with an analysis of variance (ANOVA) fol-lowed by Fisher PLSD post hoc test. P , 0.05 wasconsidered significant. For the purpose of comparingparallel experiments graphically, data were expressedas the percentage of the corresponding control (6SEM). The images were saved in Adobe PhotoShopversion 5.02 (Adobe Systems, Inc., Mountain View, CA)and Canvas version 5.03 (Deneba Software, Miami,FL) and printed on an Epson Stylus Color 900 (SeikoEpson Corp., El Segundo, CA).

RESULTS

As previously reported (9, 47), the transgenic micedisplayed profoundly abnormal motor behavior charac-terized by a moderate resting tremor, unsteady rear-ing, tic-like grooming, and a hunched posture com-

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pared to wild-type mice. These mice showed conspicu-ous feet clasping posture when suspended by theirtails. In contrast, the knock-in mice (with either 94 or71 CAG repeats) showed normal posture and groomingcompared to wild-type mice. None of the knock-in miceshowed any abnormal posture of the limbs when theywere suspended by the tail. Also in contrast to thetransgenic mice that showed pronounced weight loss at3 months of age (wild-type mice, 32.62 6 1.82 g; trans-genic mice, 15.03 6 1.07 g (P 5 0.0001) Student’s t test,n 5 7–8), the weight of the 94 CAG or 71 CAG repeatknock-in mice was not significantly different from theircorresponding controls at a similar age (wild-type mice,27.61 6 1.78 g; 71 CAG repeat mice, 27.24 6 1.79 g; 94CAG repeat mice, 27.79 6 1.29 g (NS) ANOVA, n 5

–7).

mmunolabeling for Striatal Antigens in Transgenicand Knock-In Mice

Immunohistochemistry for a broad range of antigensresent in striatal neurons was performed to deter-ine whether expression of the HD mutation dis-

upted the chemical anatomy of the striatum in theseouse models.The striatum is organized into striosomes and ma-

rix, two compartments with different inputs, outputs,nd neurochemical characteristics (30). The calciuminding protein calbindin D 28K is normally expressedn striatal neurons of the matrix compartment,hereas it is low or absent in neurons of the striosomes

27). Immunolabeling for calbindin D 28K in wild-typeice revealed the typical dense immunoreactivity inany striatal neurons contrasting with areas of mucheaker labeling characteristic of the matrix/striosomerganization of the striatum described in rats (Fig. 1A).

similar pattern of labeling was observed in bothransgenic and knock-in mice (Figs. 1B and 1C). Sim-larly, strong labeling for parvalbumin, somatostatin,nd acetylcholinesterase, which characterize parvalbu-in-containing GABA-ergic, somatostatinergic, and

holinergic striatal interneurons, respectively (40),as observed in all types of mice examined, whether orot they expressed an expanded CAG repeat (Fig. 2).As expected (31, 59), enkephalin immunoreactivityas weak in the striatum, but was very intense in thelobus pallidus (external pallidum in primates), whichontains the axon terminals of striatal enkephalinergiceurons (13, 17). No conspicuous difference in the levelnd pattern of immunostaining for this peptide wasbserved between transgenic (Fig. 3C) or knock-in miceFig. 3E) and the corresponding wild-type mice (Fig.

calbindin D 28K-positive neuronal cell bodies; arrows point to zonesof lighter calbindin D 28K staining known, in the rat, to correspondto the striosomes. Scale bar, 100mm.

FIG. 1. Calbindin D 28K immunoreactivity in striatal sections.Low-power photomicrographs of calbindin D 28K immunoreactivity instriatal sections of a wild-type mouse (A), transgenic mouse (B), and 94CAG repeat knock-in mouse (C). Arrowheads point to calbindin D28K-positive neuronal cell bodies; arrows point to zones of lighter

333STRIATAL ENKEPHALIN mRNA IN HUNTINGTON’S DISEASE MODELS

3A). Similarly, immunostaining for substance P wasintense in the substantia nigra (Figs. 3B, 3D, and 3F)of all mice examined. Together with the entopeduncu-lar nucleus, this region contains the axon terminals ofsubstance P-positive striatal efferent neurons (13, 15).Both enkephalinergic and substance P-containing stri-atal efferent neurons are GABA-ergic and mainly con-tain GAD65 (44, 49). Immunoreactivity for GAD65(Figs. 4C and 4D) was similar in the globus pallidusand substantia nigra of transgenic, knock-in (Figs. 4Eand 4F), and wild-type mice (Figs. 4A and 4B). Of note,immunolabeling for glial-acidic protein, a marker ofreactive astrocytes, was not increased in the striatum

FIG. 2. Immunohistochemistry of striatal interneurons. Low-powsomatostatin (B, E, H), and choline acetyltransferase (C, F, I) immrepeat (G, H, I) mice. Arrows point to labeled neurons. In B, E, and Hdue to thionin counterstaining. Scale bar, 50mm.

of transgenic or knock-in mice compared to wild-typeanimals (not shown). Because of the abundance of nu-clear inclusions in neurons of the striatum and cere-bral cortex in the transgenic mice, we have performedin situ nick translation (8), a sensitive technique todetect the presence of DNA damage (11, 18, 29) inthese mice. The autoradiographic signal was similarlyweak and diffuse in the striatum and cerebral cortex ofthe R6/2 transgenic mice (Fig. 5B) and control mice(Fig. 5A). Examination with high-magnification lightmicroscopy confirmed the absence of specifically la-beled cells in the striatum of the transgenic mice, al-though labeled cells were detected in the subventricu-

hotomicrographs from sections processed for parvalbumin (A, D, G),reactivity in wild-type (A, B, C), transgenic (D, E, F), and 94 CAGte the presence of weakly stained nuclei of immunonegative neurons

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334 MENALLED ET AL.

lar zone (Fig. 5D) as in controls (not shown), indicatingthat the absence of labeling in the striatum was notdue to a technical failure.

Levels of mRNA in the Striatum of Transgenic andKnock-In Mice

Despite the preservation of striatal architecture andthe lack of massive neuronal death indicated by theresults of the immunohistochemical studies, it is pos-sible that the neurons expressing the HD mutation arefunctionally altered. To test this hypothesis, we haveexamined the level of expression of mRNAs encodingGAD65, GAD67, enkephalin, and substance P, allmRNAs that are expressed by medium-sized spiny stri-atal neurons (7, 13). As indicated earlier, these neu-rons comprise the very large majority of striatal neu-rons and are particularly vulnerable to HD (63).

FIG. 3. Enkephalin immunoreactivity in the globus pallidus animmunohistochemistry in sagittal sections of the globus pallidus (GPSubstance P immunoreactivity in the substantia nigra (SN) of a wiabsence of significant differences in immunostaining between the di

Microscopic examination of emulsion-coated slidesprocessed for in situ hybridization histochemistry with35S-radiolabeled RNA probes complementary to eitherGAD65 or GAD67 (not shown) or substance P (Fig. 6)did not reveal any conspicuous differences in the levelor pattern of labeling in either the transgenic (R6/2line) mice or the knock-in (94 CAG repeats) mice com-pared to their respective controls. In contrast, autora-diographic signal for enkephalin mRNA, although ro-bust in wild-type (Figs. 7A, 7B, and 8A) and 71 CAGknock-in mice (not shown), was virtually absent in thetransgenic (Figs. 7C and 8B) and very reduced in theknock-in mice with 94 CAG repeats (Fig. 7D).

The selective decrease of enkephalin mRNA levels intransgenic and 94 CAG repeats mice was confirmed byquantitative analysis of film autoradiograms. Confirm-ing the qualitative observations on emulsion-coated

ubstance P immunoreactivity in the substantia nigra. Enkephalinf a wild-type (A), transgenic (C), and 94 repeat knock-in (E) mouse.ype (B), transgenic (D), and 94 CAG knock-in ((F) mouse. Note theent types of mice. Scale bar, 100mm.

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335STRIATAL ENKEPHALIN mRNA IN HUNTINGTON’S DISEASE MODELS

slides, transgenic mice had a marked decrease (84%) inenkephalin gene expression in the dorsolateral stria-tum compared to control mice (Fig. 9A). The slightdecrease of substance P in the transgenic mice was notsignificant compared to the controls. Similarly, the lev-els of GAD67 and GAD65 mRNAs in the transgenicmice were not significantly different from the levels incontrol mice. The surface area of the striatum wassimilar in transgenic and control mice (0.035 6 0.003cm2, n 5 7, versus 0.039 6 0.002 cm2, n 5 6, mean 6SEM).

A significant decrease in the level of enkephalinmRNA was also observed in the 94 CAG repeat micecompared to the 71 CAG repeat knock-in (58%) andwild-type mice (47%) (Fig. 9B). Surprisingly, the levelof substance P was significantly increased in the stri-atum of mice with 71 CAG repeats (Fig. 9B). This effectwas not due to a shrinkage of the striatum as striatal

FIG. 4. GAD65 immunoreactivity in the substantia nigra and g(SN) (A, C, E) and globus pallidus (GP) (B, D, F) of a wild-type (A, B)absence of conspicuous differences in the level of immunostaining b

surface areas were similar in the three groups of mice(wild-type, 0.046 6 0.003 cm2, n 5 6; 71 CAG, 0.042 60.003 cm2, n 5 7; 94 CAG, 0.049 6 0.003 cm2, n 5 8,mean 6 SEM). The marked decrease in enkephalinmRNA in 94 CAG repeat mice was confirmed in aseparate experiment in mice from different litters, in-dicating that the effect existed in three different littersof 94 CAG repeat mice. In this latter cohort, however,71 CAG repeat mice also showed a smaller but signif-icant decrease (37%) compared to control mice.

DISCUSSION

The striatum is the primary site of neuronal death inHD but brain imaging studies of presymptomatic genecarriers or patients at early stages of the disease showno or limited striatal atrophy (34, 43, 50, 66). Thisindicates that striatal neurons may tolerate the pres-

us pallidus. GAD65 immunohistochemistry in the substantia nigraansgenic (C, D), and 94 CAG repeat knock-in (D, F) mouse. Note theeen the various mice. Scale bar, 100mm.

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336 MENALLED ET AL.

ence of moderate expansion of CAG repeats in hunting-tin for many years without dying. However, decreasedglucose metabolism (43) and increased lactate produc-tion (38) suggest that surviving striatal neurons maybe dysfunctional prior to cell death.

The molecular effects of the HD mutation prior tocell death have been difficult to determine because fewbrains come to autopsy at an early stage of the disease.Mouse models of the disease are now providing aunique opportunity to address this question. In the

FIG. 5. In situ nick translation in wild-type and transgenic micewild-type (A) and a transgenic (B) mouse that were processed for dX, cerebral cortex; STR, striatum; arrows point to the subventricuright-field photomicrographs of ISNT signal in the striatum (C) anpecific labeling in the striatum despite the presence of numerousabeled cells (arrow) as a positive control. Scale bar: A, B (shown in

present study, we report that enkephalin mRNA ismarkedly decreased in two very different mouse mod-els of HD prior to cell death and clear alterations in thechemical architecture of the striatum.

Transgenic versus Knock-In Mice

Our results in the R6/2 transgenic mice confirmedprevious data indicating that behavioral anomalies oc-curred prior to neuronal death in these animals (9, 16,

and B) Low-power photomicrographs of sections of the striatum oftion of DNA strand breaks by ISNT and emulsion autoradiography.zone (SVZ) near the area illustrated in D. (C and D) High-power

ubventricular zone (D) of a transgenic mouse. Note the absence ofrons (one at arrowhead) although the SVZ contains conspicuously200mm; C, D (shown in D), 10mm.

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337STRIATAL ENKEPHALIN mRNA IN HUNTINGTON’S DISEASE MODELS

47). This is consistent with our observation that immu-nostaining for calbindin D 28K and strong immunore-activity for peptides present in striatal efferent neu-rons are preserved in these mice. These transgenicmice have prominent nuclear aggregates containingthe transgene as well as ubiquitin (16, 45, 46). Similaraggregates are present in postmortem human brain,particularly in juvenile onset cases (20).

Like other lines of mice with a knock-in CAG repeatexpansion (58, 64), the knock-in mice used in our studydid not display overt motor anomalies similar to thoseseen in the transgenic mice at the same age. Westernblots and immunohistochemical studies of brains ofknock-in mice showed that the level of expression ofthe mutant huntingtin is similar to that of normalhuntingtin in wild-type mice (Wu et al., in preparation,45, 46). Therefore, the absence of behavioral phenotypeis not related to the absence of expression of mutatedhuntingtin. Despite these major differences in behav-ioral phenotype, the two types of mice carrying the HDmutation examined in this study share a similar alter-

FIG. 6. Substance P mRNA in the striatum of mouse models oneurons labeled with substance P mRNA probe in the dorsolateracorresponding to either the transgenic (A) or the 94 repeat knock-in mmouse (D). Sections from each mouse model of HD and its correspondemulsion autoradiography in parallel. Scale bar, 100mm.

ation in the level of expression of enkephalin mRNA instriatal neurons.

Striatal mRNA Expression in Mouse Models of HD

The decrease in enkephalin mRNA occurred inde-pendent of changes in other mRNAs expressed by stri-atal neurons, such as substance P, GAD65, andGAD67, and was reproduced in tissue from differentlitters. Therefore, it is very unlikely that the effect wasrelated to a technical failure. The presence of a de-crease in enkephalin mRNA in both types of micestrongly suggests that this effect is due to the expres-sion of the expanded CAG repeats in huntingtin. Ourfinding that the decrease in enkephalin mRNA wasmore pronounced in the mice carrying 94 CAG repeatsthan in those carrying 71 CAG repeats indicates that,like in humans (62), the length of the CAG repeats is adetermining factor in this effect. The fact that thedecrease in enkephalin was not consistently observedin mice with 71 repeats may reflect a threshold effect

D and wild-type mice. Low-power dark-field photomicrographs oftriatum (white arrows). Labeling in sections from wild-type mice(B), from a transgenic mouse (C), and from a 94 CAG repeat knock-incontrol were processed for in situ hybridization histochemistry and

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338 MENALLED ET AL.

similar to that seen in humans with moderate repeatexpansion (36–39 CAG repeats) who do not alwaysdevelop the disease (62).

Preliminary data indicate that at 3.5 month of age,i.e., when we observed a decrease in enkephalin mRNAin the striatum, moderate nuclear staining for hun-tingtin and weak aggregates are present in striatalneurons (Li et al., unpublished observation). These ag-gregates intensify with age but never form nuclearinclusions similar to those observed in the transgenicmice (16). These data indicate that the mutated hun-tingtin expressed in the knock-in mice can form aggre-gates despite the presence of an arginine in position 42.Furthermore, they suggest that abnormal cellular lo-calization and/or aggregation of the mutated proteinmay contribute to the cellular phenotype we have ob-served in the knock-in mice. Finally, the data add togrowing evidence that typical nuclear inclusions arenot required for neuronal dysfunction in HD (57, 60).

FIG. 7. Enkephalin mRNA in the striatum of mouse models oeurons labeled with an enkephalin mRNA probe in the dorsolaterabeling in sections from wild-type mice corresponding to either the4 CAG repeat mouse (D). Note that in A and B, neurons are denseirtual absence of silver grains over individual neurons in (C) and thecale bar, 100mm.

Enkephalinergic neurons of the striatum project pri-marily to the external pallidum, whereas the maintargets of striatal efferent neurons containing sub-stance P are the internal pallidum and the substantianigra (28, 31, 49). In presymptomatic gene carriers andpatients with early stage, adult-onset HD, immunore-activity for enkephalin decreased earlier in the exter-nal pallidum than that for substance P in the internalpallidum (2, 51, 56). Although this difference does notapply to the substantia nigra, these data led to thehypothesis that enkephalinergic neurons were morevulnerable to the disease process than substanceP-containing neurons (51). This was further supportedby studies of cannabinoid receptors, which are locatedpresynaptically on axon terminals of striatal outputneurons in both pallidal segments (53), and by an ear-lier loss of enkephalin than of substance P mRNA inthe striatum of symptomatic patients at early stages ofthe disease (54). A previous study from our laboratory

D and wild-type mice. Low-power dark-field photomicrographs oftriatum of mouse models of HD and corresponding wild-type mice.nsgenic (A) or the knock-in mice (B), a transgenic mouse (C), and alabeled for enkephalin mRNA (white arrows), contrasting with thecrease in the number of silver grains over individual neurons in (D).

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339STRIATAL ENKEPHALIN mRNA IN HUNTINGTON’S DISEASE MODELS

did not find evidence of decreased enkephalinergicmRNA in the striatum of two presymptomatic patients

ith decreased enkephalin immunoreactivity in thexternal pallidum, suggesting that a period of neuronalysfunction preceded neuronal death (1).The present study also supports the hypothesis of a

rotracted period of neuronal dysfunction prior to celleath in mouse models of HD. Indeed, the decrease innkephalin mRNA expression observed in our studyas clearly not related to a massive death of enkepha-

inergic neurons. Analysis of emulsion-dipped sectionshowed that neuronal density was not decreased inarallel with the loss of labeling for enkephalin mRNA.urthermore, axon terminals expressing enkephalin

mmunoreactivity were at least as intense in the globusallidus of both transgenic and knock-in mice as inild-type mice. Finally, mRNAs for GAD65 andAD67, which are expressed in striatal enkephalin-

FIG. 8. Enkephalin mRNA in the striatum of mouse models ofHD and wild-type mice. High-power bright-field photomicrographs ofneurons labeled for enkephalin mRNA in the striatum of a trans-genic (B) and a corresponding wild-type mouse (A). Labeled neurons(arrows) are characterized by the presence of dense clusters of silvergrains overlying cells counterstained with hematoxylin–eosin. Notethe absence of enkephalin-labeled cells in (B) despite the normalneuron density. Scale bar, 10mm.

rgic neurons, were not decreased in the striatum ofither mutant mouse, a result compatible with the lackf decrease in striatal GABA up to at least 12 weeks ofge in the transgenic mouse (52). Although a compen-atory increase in GAD mRNA in spared neurons can-ot be ruled out, the data suggest a selective alterationf a subset of mRNAs in enkephalinergic neurons. Thiss reminiscent of previous observations that mRNAsncoding dopamine and the mGLUR1 metabotropiclutamate receptors, but not other subtypes of gluta-ate receptors, are selectively decreased in the trans-

enic mice (10).The dissociation between very low levels of enkepha-

in mRNA in the striatum and strong enkephalin im-unoreactivity in the globus pallidus was unexpected.

ndeed, in humans, marked decreases in enkephalinmmunolabeling have been observed in the externalallidum early in the disease and even in presymptom-tic gene carriers (51). The immunoreactive fibers ob-erved in mutant mice had the characteristic morphol-gy of striatopallidal axons (31), which provide theulk of enkephalin to the globus pallidus (17). There-ore, it is unlikely that the enkephalinergic immunore-ctivity observed in our study originates from the smallopulation of enkephalinergic neurons intrinsic to thelobus pallidus (35). The dissociation between en-ephalin in globus pallidus and enkephalin mRNA inhe striatum could reflect an impairment of neuropep-ide release because huntingtin, which is associatedith synaptic vesicles and microtubules, could play a

ole in vesicular release (19, 61).

ignificance of Findings in the Mouse Models

Although decreases in striatal enkephalin mRNAere not found in presymptomatic gene carriers (1),umerous studies have documented a decrease in stri-tal enkephalin mRNA levels early after symptomsnset (54). Similarly, after local administration of theMDA agonist quinolinic acid into the striatum, en-ephalin mRNA levels decreased as early as 6 h postin-

ection (8). As observed in the mouse, this decreaseccurred in the absence of neuronal loss, without sig-ificant decrease in enkephalin immunoreactivity inhe globus pallidus, and precedes the occurrence ofNA damage detectable by in situ nick translation andmulsion autoradiography (8). The similarity betweenhis effect of quinolinic acid and our observations in thewo mouse models is of interest for several reasons.irst, local injections of quinolinic acid into the stria-um eventually induce, at later times, a pattern ofeuronal death similar to that observed in HD, leadingo the hypothesis of a role for excitotoxicity in HD (6,5). Second, recent studies have demonstrated thatnother anomaly shared by the two types of transgenicice examined in this study is an increased sensitivity

f striatal and cortical neurons to the stimulation of

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340 MENALLED ET AL.

NMDA receptors (45, 46). Taken together, these obser-vations raise the possibility that the decrease in en-kephalin mRNA observed in mouse models of HD maybe due to an increased cellular response to endogenousglutamate in vivo.

In conclusion, a decrease in enkephalin mRNA withreservation of other mRNAs present in the sameGAD65 and GAD67) or neighboring neurons (GAD65,AD67, and substance P) occurred at early stage ofisease in two mouse models of HD. This may providen early indicator of neuronal dysfunction in a sub-opulation of striatal neurons known to be particularlyulnerable to the disease. Monitoring the effects ofotential treatments on decreases in striatal enkepha-in mRNA in the knock-in mice could provide insightsnto the beneficial effects of drugs at early, even pre-ymptomatic, stages of the disease.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service Grant MH44894 and the Hereditary Disease Foundation. We are grateful toDr. M. Levine (UCLA) for helpful discussions and to Mr. M. Naza-rinia for help with the manuscript and illustrations.

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